Non-Anaphylactogenic IgE Fusion Proteins

ABSTRACT

The present invention provides compositions and methods for the use of antigenic peptides derived from the Fc portion of the epsilon heavy chain of IgE molecules from two unrelated species as vaccines for the treatment and prevention of IgE-mediated allergic disorders, in particular, the invention provides compositions for the treatment and prevention of IgE-mediated allergic disorders comprising an immunogenic amount of one or more antigenic peptides.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for the use ofantigenic peptides derived from the Fc portion of the epsilon heavychain of IgE molecules as vaccines for the treatment and prevention ofIgE-mediated allergic disorders. In particular, the present inventionrelates to compositions comprising at least one antigenic peptidecomprising an amino acid sequence derived from the CH3 domain of IgEmolecules from two different species for the treatment or prevention ofan IgE-mediated allergic disorder. The present invention also relates tocompositions comprising antigenic peptides coupled to heterologouscarrier proteins and optionally further comprising an adjuvant. Thecompositions of the present invention induce anti-IgE antibodies whichbind to soluble (free) IgE in serum and other bodily fluids, prevent IgEfrom binding to its high affinity receptors on mast cells and basophils,and do not cross-link receptor-bound IgE. The present invention furtherrelates to methods of administering compositions of the invention toanimals, preferably mammals and most preferably humans, for thetreatment or prevention of IgE-mediated allergic disorders.

BACKGROUND OF THE INVENTION

Immune-mediated allergic (hypersensitivity) reactions are classifiedinto four types (I-IV) according to the underlying mechanisms leading tothe expression of the allergic symptoms. Type I allergic reactions arecharacterized by IgE-mediated release of vasoactive substances such ashistamine from mast cells and basophils. The release of these substancesand the subsequent manifestation of allergic symptoms are initiated bythe cross-linking of allergen-bound IgE to its receptor on the surfaceof mast cells and basophils.

An IgE antibody is a complex molecule consisting of two identical heavychains and two identical light chains held together by disulfide bondsin a “Y” shape-configuration. Each light chain consists of a variable(V_(L)) domain linked to a constant domain (C_(L)), and each heavy chainconsists of a variable domain (V_(H)) and four constant domains (CH1,CH2, CH3, and CH₄, also known as Cε1, Cε2, Cε3, and Cε4; respectively).The two arms of an IgE antibody contain the site at which an IgEantibody binds to its specific antigen (allergen) and each arm isreferred to as a Fab (fragment-antigen-binding) fragment. The tail of anIgE antibody is termed Fc (fragment-crystalline) as it can form crystalswhen separated from the Fab fragments of the antibody under appropriateexperimental conditions. The Fc fragment of an IgE antibody consists ofthe CH2, CH3, and CH4 domains and contains the biologically activestructures of the IgE antibody (e.g., receptor binding sites).

The production of IgE antibodies requires interactions andcollaborations among three cells; antigen presenting ceils (APC), Tlymphocytes (T helper cells; Th) and antibody producing cells (Blymphocytes; B cells). When a foreign substance, an allergen, isintroduced for the first time into the body of subjects (e.g., byinhalation of environmental allergen, ingestion of certain foods, or viathe skin), the allergen is taken up by APC's (e.g., macrophages) whichthen digest or process the allergen into smaller fragments (epitopes).These fragments are displayed on the surface of APC's in associationwith specific molecules known as major histocompatibility complexproteins. The allergen fragment/MHC complex displayed on the surface ofAPC's is recognized and bound by receptors on the surface of specific Tlymphocytes. This recognition and binding event leads to the activationof T lymphocytes and the subsequent expression and secretion ofcytokines such as interleukin-4(IL-4). These cytokines induce themultiplication, clonal expansion and differentiation of B cells specificfor the allergen in question (i.e., B cell which express on theirsurface immunoglobulin receptors capable of binding to the allergen) andultimately lead to the production of IgE antibodies from these B cells.A portion of the activated T lymphocytes and IgE producing B cellseventually become committed to a pool of cells called T and B memorycells, which are capable of faster recognition of allergen uponsubsequent exposure to the allergen.

In individuals suffering from type I allergic reactions, exposure to anallergen for a second time leads to the production of high levels of IgEantibodies specific for the allergen as a result of the involvement ofmemory B and T cells in the 3-cell interaction required for IgEproduction. The high levels of IgE antibodies produced cause an increasein the cross-linking of IgE receptors on mast cells and basophils byallergen-bound IgE, which in turn leads to the activation of these cellsand the release of the pharmacological mediators that are responsiblefor the clinical manifestations of typo I allergic diseases.

Two receptors with differing affinities for IgE have been identified andcharacterized. The high affinity receptor (FcεRI) is expressed on thesurface of mast cells and basophils. The low affinity receptor(FcεRII/CD23) is expressed on many cell types including B cells, Tcells, macrophages, eosinophils and Langerhan cells. The high affinityIgE receptor consists of three subunits (alpha, beta and gamma chains).Several studies demonstrate that only the alpha chain is involved in thebinding of IgE, whereas the beta and gamma chains (which are eithertransmembrane or cytoplasmic proteins) are required for signaltransduction events. The identification of IgE structures required forIgE to bind to the FcεRI on mast cells and basophils is of utmostimportance in devising strategies for treatment or prevention ofIgE-mediated allergies. For example, the elucidation of the IgEreceptor-binding site could lead to the identification of peptides orsmall molecules that block the binding of IgE to receptor-bearing cellsin vivo.

Over the last 15 years, a variety of approaches have been utilized todetermine the FcεRI binding site on IgE. These approaches can beclassified into five different categories. In one approach, smallpeptides corresponding to portions of the Fc part of an IgE moleculewere produced and analyzed for their ability to inhibit IgE from itsreceptors. See, for example, Nakamura et al., EP0263655 published Apr.13, 1988, Burt et al., 1987, European Journal of Immunol., 17:437-440;Helm et al., 1988, Nature 331:180-183; Helm et al., 1989, PNAS86:9465-9469; Vercelli et al., 1989, Nature 338:649-651; Nio et al.,1990, Peptide Chemistry. 2: 203-208; Nio et al., 1993, FEBS Lett.319:225-228; and Nio et al., 1992, FEBS Lett. 314:229-231. Although manyof the peptides described in these studies were shown to inhibit thebinding of IgE to its receptors, different studies reported differentsequences as being responsible for IgE binding.

Helm et al. (1988, Nature 331:180-183) identified a 75 amino acidpeptide that spans the junction between CH2 and CH3 domains of IgE andshowed that this peptide binds to the IgE receptor with an affinityclose to that of the native IgE molecule. On the other hand, Basu et al.(1993, Journal of Biological Chemistry 268: 13118-13127) expressedvarious fragments from IgE molecules and found that only those fragmentscontaining both the CH3 and CH4domains were able to bind IgE and thatCH2 domain is not necessary for binding. Vangelista et al. (1999,Journal of Clinical Investigation 103:1571-1578) expressed only the CH3domain of IgE and showed that this domain alone could bind to IgEreceptor and prevent binding of IgE to its receptor. The results of Basuet al. and Vangelista et al. are inconsistent and conflict with those ofHelm et al. cited above.

In a second approach to identify the FcεRI binding site on IgE,polyclonal antibodies against peptides corresponding to parts of the CH2domain, CH3 domain or CH4 domain were produced and used to probe forreceptor binding site on IgE (Robertson et al., 1988, Molecular Immunol.25:103-118). Robertson et al. concluded that the amino acid residuesdefined by a peptide derived from the CH4 domain were not likely to beinvolved in receptor binding, whereas amino acid residues defined by apeptide derived from the CH3 domain of IgE were most likely proximal tothe IgE receptor-binding site (amino acids 387-401). However, theanti-CH3 peptide antibodies induced in response to the CH3 peptidereleased histamine from IgE-loaded mast cells indicating that the aminoacids defined by the CH3peptide did not define the bona fide IgEreceptor-binding site and that anti-CH3 peptide antibodies could causeanaphylaxis.

In a third approach to identify the FcεRI binding site on IgE, severalinvestigators produced IgE mutants in an attempt to identify the aminoacid residues involved in receptor binding (see, e.g., Schwarzbaum etal., 1989, European Journal of Immunology 19:1015-1023; Weetall et al.,1990, Journal of Immunology 145:3849-3854; and Presta et al., 1994,Journal of Biological Chemistry 269:26368-26373). Schwartzbaum et al.demonstrated that an IgE antibody with the point mutation proline tohistidine at amino acid residue 442 in the CH4 domain has a two foldreduced affinity for the IgE receptor. Schwartzbaum et al. concludedthat the CH4 domain of an IgE antibody is involved in IgE binding to itsreceptor. However, Schwartzbaum's conclusion contradict Weetall et al.'sconclusion that the binding of IgE to its high affinity receptorinvolves portions of the CH2 and CH3 domains of the IgE antibody, butnot the CH4 domain. Further, Schwartzbaum et al.'s conclusionscontradict Presta et al.'s conclusion that the amino acid residues ofthe IgE antibody important for binding to the FcεRI are located in theCH3 domain.

In a fourth approach to identify the FcεRI binding site on IgE, chimericIgE molecules were constructed and analyzed for their ability to bind tothe FcεRI. Weetall et al., supra constructed a series of chimeric murineIgE-human IgG molecules and tested their binding to the IgE receptor.Weetall et al., supra concluded that the CH4 domain does not participatein receptor binding and that the CH2 and CH3 domains are both requiredfor binding to the high affinity receptor on mast cells. In anotherstudy, Nissim et al. (1993, Journal of Immunol 150:1365-1374) tested theability of a series of human IgE-murine IgE chimera to bind to the FcεRIand concluded that only the CH3 domain is needed for binding to theFcεRI. The conclusion by Nissim et al. corroborates the conclusion byVangelista et al. that the CH3domain of IgE alone binds to the FcεRI.However, the conclusions by Nissim et al. and Vangelista et al.contradict the conclusions of Weetall et al. and Robertson et al.

Presta et al., supra produced chimeric human IgG in which the CγH2 wasreplaced with CH3 from human IgE. When tested for receptor binding, thischimera bound to the FcεRI albeit with a four-fold reduced affinitycompared with native IgE. The results of Presta et al. appear tocorroborate with the results of Nissim et al., but conflict with thoseof Weetall et al., Helm et at., and Basu et. al., cited above. In afurther attempt to define the exact amino acid residues responsible forthe binding of IgE to its receptor, Presta et al. inserted specificamino acid residues corresponding to CH2-CH3 hinge region and threeloops from the CH3 domain of human IgE into their analogous locationswithin human IgG and called these mutants IgGEL. Unfortunately, whenthese IgGEL variants were tested for receptor binding, they exhibitedminimal binding compared to the native IgE or the IgG in which the fulllength IgE CH3 domain replaced the full length CγH2 domain. In a fifthapproach to identify the FcεRI binding site on IgE, monoclonalantibodies have been developed and analyzed for their ability to blockIgE binding to the FcεRI. See, for example. Del Prado et al., 1991.Molecular Immunology 28:839-844; Keegan et al., 1991, MolecularImmunology 28:1149-1154; Hook et al., 1991. Molecular Immunology28:631-639; Takemoto et al., 1994, Microbiology and Immunology 38:63-71;and Baniyash et al., 1988, Molecular immunology 25:705-711. Althoughmany monoclonal antibodies have been developed, they have providedlittle information on the bona fide IgE receptor-binding site because inmany cases the amino acid sequence recognized by these monoclonalantibodies have not or could not be identified. Further, the monoclonalantibodies developed may block IgE from binding to its receptor bysteric hindrance or induction of severe conformational changes in theIgE molecule, rather than by the binding and masking of IgE residuesdirectly involved in receptor binding.

It is apparent from the above discussion that approaches that have beendevised to identify the receptor binding site on IgE have producedconflicting results. The difficulty in the identification of the aminoacid residues of IgE responsible for receptor binding could be furthercomplicated by the possibility that the site on IgE used for binding tothe receptor may not be a linear sequence of amino acids, which could bemimicked by a synthetic peptide. Rather, the binding site may be aconformational determinant formed by multiple amino acids that are farapart in the IgE protein sequence which are brought into close proximityonly in the native three-dimensional structure of IgE. Studies with IgEvariants, IgE chimera, and monoclonal anti-IgE antibodies stronglysuggest that the binding site is a conformational determinant.

Currently, IgE-mediated allergic reactions are treated with drugs suchas antihistamines and corticosteroids which attempt to alleviate thesymptoms associated with allergic reactions by counteracting the effectsof the vasoactive substances released from mast cells and basophils.High doses of antihistamines and corticosteroids have deleterious sideeffects such as renal and gastrointestinal toicities. Thus, othermethods for treating type I allergic reactions are needed.

One approach to the treatment of type I allergic disorders has been theproduction of monoclonal antibodies which react with soluble (free) IgEin serum, block IgE from binding to its receptor on mast cells andbasophils, and do not bind to receptor-bound IgE (i.e., they arenon-anaphylactogenic). Two such monoclonal antibodies (rhuMab E25 andCGP56901) are in advanced stages of clinical development for treatmentof IgE-mediated allergic reactions (see. e.g., Chang, T. W., 2000,Nature Biotechnology 18:157-62). The identity of the amino acid residuesof the IgE molecule recognized by these monoclonal antibodies are notknown and it is presumed that these monoclonal antibodies recognizeconformational determinants on IgE.

Although early results from clinical trials with therapeutic anti-IgEmonoclonal antibodies suggest that these therapies arc effective in thetreatment of atopic allergies, the use of monoclonal antibodies forlong-term treatment of allergies has some significant shortcomings.First, since these monoclonal antibodies were originally produced inmice, they had to be reengineered so as to replace mouse sequences withconsensus human IgG sequences (Presta et al., 1993, The Journal ofImmunology 151:2623-2632). Although this “humanization” process has ledto production of monoclonal antibodies that contain 95% human sequences,there remain some sequences of mouse origin. Since therapy with theseanti-IgE antibodies requires frequent administration of the antibodiesover a long period of time, some treated allergic patients could producean antibody response against the mouse sequences that still remainwithin these therapeutic antibodies. The induction of antibodies againstthe therapeutic anti-IgE would negate the therapeutic impact of theseanti-IgE antibodies at least in some patients. Second, the cost oftreatment with these antibodies will be very high since high doses ofthese monoclonal antibodies are required to induce a therapeutic effect.Moreover, the frequency and administration routes with which theseantibodies have to be administered are inconvenient. A more attractivestrategy for the treatment of IgE-mediated disorders is theadministration of peptides which induce the production of anti-IgEantibodies.

One of the most promising treatments for IgE-mediated allergic reactionsis the active immunization against appropriate non-anaphylactogenicepitopes on endogenous IgE. Stanworth et al. (U.S. Pat. No. 5,601,821)described a strategy involving the use of a peptide derived from the CH4domain of the human IgE coupled to a heterologous carrier protein as anallergy vaccine. However, this peptide has been shown not to induce theproduction of antibodies that react with native soluble IgE. Further,Hellman (U.S. Pat. No. 5,653,980) proposed anti-IgE vaccine compositionsbased on fusion of full length CH2-CH3domains (approximately 220 aminoacid long) to a foreign carrier protein. However, the antibodies inducedby the anti-IgE vaccine compositions proposed in Hellman will mostlikely result in anaphylaxis since antibodies against some portions ofthe CH2 and CH3 domains of the IgE molecule have been shown tocross-link the IgE receptor on the surface of mast cell and basophilsand lead to production of mediators of anaphylaxis (see, e.g., Stadleret al., 1993, Int. Arch. Allergy and Immunology 102:121-126). Therefore,a need remains for vaccines for the treatment of IgE-mediated allergicreactions which do not induce anaphylactic antibodies.

The significant concern over Induction of anaphylaxis has resulted inthe development of another approach to the treatment of type I allergicdisorders consisting of mimotopes that could induce the production ofanti-IgE polyclonal antibodies when administered to animals (see, e.g.,Rudolf, et al., 1998, Journal of Immunology 160:3315-3321). Kricek etat. (International Publication No. WO 97/31948) screened phage-displayedpeptide libraries with the monoclonal antibody BSWI7 to identify peptidemimotopes that could mimic the conformation of the IgE receptor binding.These mimotopes could presumably be used to induce polyclonal antibodiesthat react with free native IgE, but not with receptor-bound IgE as weltas block IgE from binding to its receptor. Kricek et al. disclosedpeptide mimotopes that are not homologous to any part of the IgEmolecule and are thus different from peptides disclosed in the presentinvention.

A major obstacle facing the development of an anti-IgE vaccine is thelack of information regarding the precise amino acids representingnon-anaphylactogenic IgE determinants that could be safely used toimmunize allergic subjects and induce non-anaphylactogenic polyclonalantibodies (i.e., polyclonal anti-IgE antibodies that do not bind toreceptor-bound IgE). The peptide compositions of the present inventionare selected to be non-anaphylactogenic; i.e., the peptide compositionsdo not result in production of anti-IgE antibodies that could bind orcause cross-linking of IgE bound to mast cells or basophils. Thuspeptides of the present invention have superior safety profile and aredifferentiated by sequence composition from disclosed vaccines based onfull-length C2H-CH3 domains.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for the use ofantigenic peptides derived from the Fc portion of the epsilon heavychain of IgE molecules as vaccines for the treatment and prevention ofIgE-mediated allergic disorders. In one embodiment, the inventionprovides compositions for the treatment and prevention of IgE-mediatedallergic disorders comprising an immunogenic amount of one or moreantigenic peptides derived from the CH3 domains of IgE molecules fromtwo unrelated species effective for treatment or prevention of anIgE-mediated allergic disorder. Preferably, compositions of the presentinvention comprise an immunogenic amount of one or more antigenicpeptides comprising the amino acid sequence of SEQ ID NOS: 2, 3, 10, 11,12, 13 or 14 or an antigenic fragment, derivative or variant thereof.

The antigenic peptides can be supplied by direct administration orindirectly as “pro-drugs” using somatic cell gene therapy.

In a preferred embodiment, the present invention is based, in part, onthe discovery that antigenic peptides comprising conserved amino acidresidues of the CH3 domain of an IgE molecule from one species flankedby variable amino acid residues of the CH3 domain of an IgE moleculefrom a second unrelated species are capable of inducing a high titer ofanti-IgE antibodies when administered to an animal without causinganaphylaxis. The Applicants compared the primary amino acid sequences ofIgE molecules from different species, e.g., rat IgE and dog IgE, andidentified conserved amino acid residues in the CH3 domains of the IgEmolecules from the different species. The Applicants also determinedthat the conserved amino acid residues in the CH3 domains of IgEmolecules from different species are flanked by amino acid residues thatvary from species to species (referred to as “the variable amino acidresidues”).

Accordingly, in one embodiment, the present invention encompassesantigenic peptides comprising amino acid residues of the CH3 domain ofan IgE molecule from a first species flanked by amino acid residues ofthe CH3 domain of an IgE molecule from a second unrelated species. Theamino acid residues of the CH3 domain of the IgE molecule from the firstspecies, which comprise the antigenic peptide, are conserved in the CH3domain of the IgE molecule of the second unrelated species. However, theamino acid residues of the CH3domain of the IgE molecule of the secondunrelated species which comprise the antigenic peptide are not conserved(i.e., vary) in the CH3 domain of the IgE molecule of the first species.Thus, for example, an antigenic peptide of the present invention couldcomprise conserved amino acid residues of the CH3 domain of the canineIgE molecule flanked by amino acid residues of the CH3 domain of the ratIgE molecule. Such an antigenic peptide would preferably be administeredto a dog to treat or prevent an IgE-mediated allergic disorder. Thepresent invention further provides antigenic fusion proteins derivedfrom a single species, which do not cause anaphylaxis when administeredto an animal. Preferably, such an antigenic fusion protein having thesequence SEQ ID NO: 27.

The present invention also provides pharmaceutical compositionscomprising an immunogenically effective amount of one or more antigenicpeptides derived from the CH3domains of IgE molecules from the same orfrom two or more unrelated species and one or more pharmaceuticalacceptable carriers. In one embodiment, a pharmaceutical composition ofthe invention comprises an immunogenically effective amount of one ormore antigenic peptides derived from the CH3 domains of IgE moleculesfrom two unrelated species and one or more pharmaceutically acceptablecarriers. In another embodiment, a pharmaceutical composition of theinvention comprises one or more pharmaceutical carriers and animmunogenically effective amount of one or more antigenic peptidederived from the CH3 domains of IgE molecules from two unrelated species(SEQ ID NOS: 2, 3, and 10-14) and a heterologous carrier protein such asSEQ ID NOS: 9 and 23.

The present invention also provides pharmaceutical compositionscomprising an immunogenically effective amount of one or more antigenicpeptides derived from the CH3domains of IgE molecules from two unrelatedspecies, a pharmaceutically acceptable carrier, and an adjuvant.Adjuvants encompass any compound capable of enhancing an immune responseto an antigen. Examples of adjuvants which may be effective, include,but are not limited to: aluminum hydroxide, monophosphoryl lipid A(MPLA)-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine,N-acetytmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine,simple immunostimulatory oligonucleotides, cytokines such as IL-12, IL-2or IL-1, saponins, and microbial toxins such as cholera toxin, heatlabile toxin and genetically altered derivatives of them.

In another embodiment, a pharmaceutical composition of the inventioncomprises a pharmaceutical carrier, an adjuvant and an immunogenicallyeffective amount of one or more antigenic fusion proteins comprising anantigenic peptide derived from the CH3 domains of IgE molecules from twounrelated species and a heterologous carrier protein. In a preferredembodiment, a pharmaceutical composition of the invention comprises apharmaceutical carrier, an adjuvant and an immunogenically effectiveamount of one or more antigenic peptides comprising of the amino acidsequence of SEQ ID NOS: 2, 3 and 10-14.

In another preferred embodiment, a pharmaceutical composition of thepresent invention comprises a pharmaceutical carrier, an adjuvant, andan immunogenically effective amount of one or more fusion proteinscomprising the amino acid sequence of SEQ ID NOS: 2,3, and 10 to 14.

The present invention also provides methods of administeringcompositions of the invention to animals, preferably mammals and mostpreferably humans for the treatment or prevention of IgE-mediatedallergic disorders. The compositions of the present invention are insuitable formulation to be administered to animals, preferably mammalssuch as companion animals (e.g., dogs, cats, and horses) and livestock(e.g., cows and pigs), and most preferably humans. The compositions ofthe invention are administered in an amount effective to elicit animmune response, for example, the production of polyclonal antibodieswith specificity for an IgE molecule. In one embodiment, thecompositions of the invention are administered in an amount effective toinduce the production of polyclonal or monoclonal antibodies withspecificity for the Fc portion of an IgE molecule required for IgE tobind to its receptor (i.e., the CH3 domain of an IgE molecule). In apreferred embodiment, the compositions of present invention areadministered in an amount effective to induce the production of anti-IgEantibodies which bind to soluble (free) IgE in serum and other bodilyfluids, prevent IgE from binding to its high affinity receptors on mastcells and basophils, and do not cross-link receptor-bound IgE.Accordingly, the compositions of the invention are administered in anamount effective to induce the production of polyclonal antibodies whichdo not induce anaphylaxis for the treatment or prevention ofIgE-mediated allergic disorders.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Baculovirus expressed human CH3 domain separated by SDS-SAGE on4-12% gels under reducing conditions. The 11 kDA CH3 domain can be seenin lane 4. No corresponding bands were observed in the sf-9 celt control(lane 2) or in wild type baculovirus (lane 3). Positions of molecularmass standards (kDa) are indicated in lane 1.

FIG. 2. Immunoblotting of baculovirus expressed Human CH3 domain withrabbit A# 145 RBS-2 antiserum. Samples were separated by SOS-SAGE on4-12% gels under reducing conditions. The 11 kDA CH3 domain can be seenin lane 4. No bands were observed in the sf-9 celt control (lane 2) orin wild type baculovirus (lane 3). Positions of molecular mass standards(kDa) are indicated in lane 1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for the use ofantigenic peptides derived from the Fc portion of the epsilon heavychain of IgE molecules as vaccines for the treatment and prevention ofIgE-mediated allergic disorders. In particular, the present inventionprovides compositions comprising an immunogenic amount of an antigenicpeptide derived from the CH3 domains of IgE molecules from two unrelatedspecies effective for treatment or prevention of an IgE-mediatedallergic disorder. Preferably, compositions of the present inventioncomprise an immunogenic amount of one or more antigenic peptidescomprising the amino acid sequence of SEQ ID NOS: 1 to 6 and 10 to 14.

The antigenic peptides of the present invention comprise an amino acidsequence of the CH3 domains of IgE molecules from two unrelated speciesand induce the production of anti-IgE antibodies, which are notanaphylactic. In particular, the antigenic peptides of the presentinvention induce the production of anti-IgE antibodies which bind tosoluble (free) IgE in serum and other bodily fluids, prevent IgE frombinding to its high affinity receptors on mast cells and basophils, anddo not cross-link receptor-bound IgE. The antigenic peptides of thepresent invention may be coupled to one or more heterologous peptides.The antigenic peptides of the invention can be supplied by directadministration or indirectly as “pro-drugs” using somatic cell genetherapy.

In one embodiment, an antigenic peptide of the invention comprises anamino acid sequence comprising amino acid residues of the CH3 domain ofan IgE molecule from a first species flanked by amino acid residues ofthe CH3 domain of an IgE molecule from a second, preferably unrelated,species. An antigenic peptide of the invention comprises at least10amino acid residues of the CH3 domain of an IgE molecule from a firstspecies, at least 15amino acid residues of the CH3 domain of an IgEmolecule from a first species, at least 20amino acid residues of the CH3domain of an IgE molecule from a first species, or at least 25amino acidresidues of the CH3 domain of an IgE molecule from a first species.Further, an antigenic peptide of the invention comprises at least 10amino acid residues of the CH3domain of an IgE molecule from a secondspecies, at least 15 amino acid residues of the CH3domain of an IgEmolecule from a second species, at least 20 amino acid residues of theCH3domain of an IgE molecule from a second species, or at least 25 aminoacid residues of the CH3 domain of an IgE molecule from a secondspecies.

In specific embodiments, an antigenic peptide of the invention is atleast 10 amino acid residues long, at least 15 amino acid residues long,at least 20 amino acid residues long, or at least 25 amino acid residuelong, or at least 30 amino acid residues long. In a preferredembodiment, an antigenic peptide of the invention comprises an aminoacid sequence comprising amino acid residues of the CH3 domain of an IgEmolecule from a first species flanked by amino acid residues of the CH3domain of an IgE molecule from a second unrelated species and saidantigenic peptide is between 28 and 31 amino acid residues. The presentinvention also provides antigenic fusion proteins comprising anantigenic peptide and a heterologous carrier protein. In a specificembodiment, an antigenic fusion protein comprises amino acid residues ofthe CH3 domain of an IgE molecule from a first species flanked by aminoacid residues of the CH3 domain of an IgE molecule from a secondunrelated species and a heterologous protein carrier. In a preferredembodiment, an antigenic fusion protein of the present inventioncomprises the amino acid sequence of SEQ ID NOS: 2, 3, and 10 to 14.

The present invention also provides antigenic peptides or antigenicfusion proteins of the invention in which one or more amino acidsubstitutions, additions or deletions has been introduced. Mutations canbe introduced by standard techniques known to those of skill in the art.For example, one or more mutations at the nucleotide level which resultin one or more amino acid mutations can be introduced by site-directedmutagenesis or PCR-mediated mutagenesis. Preferably, conservative aminoacid substitutions are made at one or more predicted non-essential aminoacid residues. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Alternatively, mutations can be introduced randomly alongall or part of the coding sequence, such as by saturation mutagenesis,and the resultant mutants can be screened for their ability to induceanti-IgE antibodies which do not cause anaphylaxis.

The present invention also provides methods for treating or preventingIgE-mediated allergic disorders in animals, preferably mammals and mostpreferably humans, comprising administering pharmaceutical compositions,which do not induce anaphylaxis. The pharmaceutical compositions to beadministered in accordance with the methods of the present inventionencompass antigenic peptides derived from the CH3 domains of IgEmolecules from two unrelated species. The pharmaceutical compositions tobe administered in accordance with the methods of the present inventionalso include: (i) recombinant antigenic peptides having an amino acidsequence comprising amino acid residues of the CH3 domain of an IgEmolecule from a first species flanked by amino acid residues of the CH3domain of an IgE molecule from a second species; (ii) recombinantantigenic fusion proteins comprising amino acid residues of the CH3domain of an IgE molecule from a first species flanked by amino acidresidues of the CH3 domain of an IgE molecule from a second species anda heterologous carrier protein; (iii) plasmid compositions comprisingpolynucleotide encoding an anligenic peptide having an amino acidsequence comprising amino acid residues of the CH3 domain of an IgEmolecule from a first species flanked by amino acid residues of the CH3domain of an IgE molecule from a second species; and (iv) plasmidcompositions comprising polynucleotides encoding for antigenic fusionproteins comprising amino acid residues of the CH3 domain of an IgEmolecule from a first species flanked by amino acid residues of the CH3domain of an IgE molecule from a second species and a heterologousearner protein.

In one embodiment, a pharmaceutical composition of the present inventioncomprises one or more antigenic peptides having the amino acid sequencecomprising amino acid residues of the CH3 domain of an IgE molecule froma first species flanked by amino acid residues of the CH3 domain of anIgE molecule from a second species. In a preferred embodiment, apharmaceutical composition of the present invention comprises one ormore antigenic peptides between 28 and 31 amino acid residues longhaving the amino acid sequence comprising amino acid residues of the CH3domain of an IgE molecule from a first species flanked by amino acidresidues of the CH3 domain of an IgE molecule from a second unrelatedspecies. In accordance with these embodiments, the pharmaceuticalcompositions may further comprise an adjuvant.

The present invention also provides pharmaceutical compositionscomprising one or more antigenic fusion proteins. In a specificembodiment, a pharmaceutical composition of the present inventioncomprises one or more antigenic fusion proteins comprising an antigenicpeptide of the invention and a heterologous carrier protein. Inaccordance with this embodiment, the pharmaceutical composition mayfurther comprise an adjuvant.

As used herein the term “heterologous carrier protein” refers to aprotein which does not possess high homology to a protein found in thespecies that is receiving a composition of the invention and elicits animmune response. A protein possesses high homology if it is greater thanat least 75% identical, more preferably at least 85% identical or atleast 90% identical to a protein as determined by any known mathematicalalgorithm utilized for the comparison of two amino acid sequences (see,e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877; Torellis and Robottl, 1994, Comput. Appl. Biosci. 10; 3-5;and Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. 85: 2444-8).Preferably, the percent identity of two amino acid sequences isdetermined by BLAST protein searches with the XBLAST program, score=50,wordlength a 3. Examples of heterologous carrier proteins include, butare not limited to, SEQ ID NOS: 7, 8 or 9, KLH, PhoE, and rmLT.

A heterologous carrier protein can be fused to the N-terminus,C-terminus or both termini of an antigenic peptide of the invention.Antigenic fusion proteins of the invention can be produced by techniquesknown to those of skill in the art, for example, by standard recombinantDNA techniques. For example, a nucleotide sequence encoding an antigenicfusion protein can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and reamplified to generate a gene sequenceencoding an antigenic fusion protein (see, e.g., Ausubel et al., infra).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A nucleic acidencoding an antigenic peptide of the invention can be cloned into suchan expression vector such that the fusion moiety is linked in-frame tothe antigenic peptide of the invention.

In a specific embodiment, a pharmaceutical composition of the presentinvention comprises an antigenic peptide having an amino acid sequencecomprising amino acid residues of SEQ ID NOS 2, 3 and 10 to 14.

In another embodiment, a pharmaceutical composition of the presentinvention comprises an antigenic fusion protein comprising the aminoacid sequence of SEQ ID NOS: 2, 3 and 10 to 14. In accordance with theseembodiments, the pharmaceutical compositions may further comprise anadjuvant.

The pharmaceutical compositions of the present invention are in suitableformulation to be administered to animals such as companion animals(e.g., dogs and cats) and livestock (e.g., pigs, cows and horses) andhumans for the treatment or prevention of IgE-mediated allergicdisorders.

IgE mediated disorders include allergic rhinitis/hay fever, asthma,atopic dermatitis, flea allergy, food allergy and inhalant allergy.

Preferably, a pharmaceutical composition of the invention comprising anantigenic peptide of the invention is administered to the same speciesas the amino acid residues derived from the CH3 domain of an IgEmolecule of the first species to treat or prevent an IgE-mediatedallergic disorder. IgE-mediated allergic disorders include, but are notlimited to, asthma, allergic rhinitis, gastrointestinal allergies suchas food allergies, eosinophilla, and conjunctivitis. The pharmaceuticalcompositions of the invention are administered to a subject (an animal)in an amount effective for the treatment, prevention or inhibition ofIgE-mediated allergic disorders, or an amount effective for inducing ananti-IgE response that is not anaphylactic, or an amount effective forinhibiting or reducing the release of vasoactive substances such ashistamine, or an amount effective for alleviating one or more symptomsassociated with an IgE-mediated allergic disorder.

The pharmaceutical compositions of the invention can be used with anyknown method of treating IgE-mediated allergic disorders. In oneembodiment, one or more pharmaceutical compositions of the invention andone or more antihistamines are administered to an animal for thetreatment or prevention of an IgE-mediated allergic disorder. In anotherembodiment, one or more pharmaceutical compositions of the invention andone or more corticosteroids are administered to an animal for thetreatment or prevention of an IgE-mediated allergic disorder. In yetanother embodiment, one or more pharmaceutical compositions of theinvention and one or more anti-IgE monoclonal antibodies (e.g., BSW17)are administered to an animal for the treatment or prevention of anIgE-mediated allergic disorder.

The present invention encompasses polynucleotide sequences encoding theantigenic peptides (SEQ IO NOS: 2,3 and 10 to 14 ), carrier proteins(SEQ ID NOS: 7, 8and 9) or antigenic fusion proteins (SEQ ID NOS: 2,3,and 10 to 14) of the invention. The present invention provides nucleicacid molecules comprising different polynucleotide sequences due to thedegeneracy of the genetic code which encode identical antigenic peptidesand antigenic fusion proteins. The polynucleotide sequence of a CH3domain of an IgE molecule can be obtained from scientific literature,Genbank, or using cloning techniques known to those of skill in the art.In particular, the present invention encompasses polynucleotidesequences encoding human, rat and canine CH3 domain of an IgE moleculedisclosed in Genbank Accession Numbers S53497, X00923, and L36872;respectively, are incorporated herein by reference.

The present invention also encompasses antigenic fusion proteinscomprising an antigenic peptide of the invention encoded by apolynucleotide sequence from two different species and a heterologouscarrier protein encoded by a polynucleotide sequence of a differentspecies from the antigenic peptide. The polynucleotide sequence of aheterologous carrier protein can be obtained from scientific literature,Genbank, or using cloning techniques known to those of skill in the art.

The polynucleotide sequence encoding an antigenic peptide or anantigenic fusion protein of the invention can be inserted into anappropriate expression vector, i.e., a vector, which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. The necessary transcriptional and translationalsignals can also be supplied by the native IgE genes or its flankingregions. A variety of host-vector systems may be utilized to express theprotein-coding sequence. These include but are not limited to mammaliancell systems infected with virus (e.g., vaccinia virus, adenovirus,etc.); insect cell systems infected with virus (e.g., baculovirus);microorganisms such as yeast containing yeast vectors, or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining polynucleotides encoding antigenic peptides or antigenicfusion proteins, and appropriate transcriptional and translationalcontrol signals. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of the nucleic acid sequence encoding an antigenic peptide oran antigenic fusion protein of the invention may be regulated by asecond nucleic acid sequence so that the antigenic peptide or theantigenic fusion protein is expressed in a host transformed with therecombinant DNA molecule. For example, expression of an antigenicpeptide or an antigenic fusion protein of the invention may becontrolled by any promoter or enhancer element known in the art.Promoters which may be used to control the expression of an antigenicpeptide or an antigenic fusion protein of the invention include, but arenot limited to, the Cytomeglovirus (CMV) immediate early promoterregion, the SV40 early promoter region (Bernoist and Chambon, 1981,Nature 290: 304-310), the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22: 787-797),the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. USA 78: 1441-1445), the regulatory sequences of themetallothionein gene (Brinster et al., 1982, Nature 296: 39-42);prokaryotic expression vectors such as the β-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731),or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25); see also “Useful proteins from recombinant bacteria” inScientific American, 1980, 242: 74-94; plant expression vectorscomprising the nopaline synthetase promoter region (Herrera-Estrella etal., Nature 303: 209-213) or the cauliflower mosaic virus 35S RNApromoter (Gardner et al., 1981, Nucl. Acids Res. 9: 2871), and thepromoter of the photosynthetic enzyme ribulose biphosphate carboxylase(Herrera-Estrella et al., 1984, Nature 310: 115-120); promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter, and the following animal transcriptional controlregions, which exhibit tissue specificity and have been utilized intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz etal., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald,1987, Hepatotogy 7:425-515); insulin gene control region which is activein pancreatic beta cells (Hanahan, 1985, Nature 315: 115-122);immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature318: 533-538; and Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444); mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45: 485-495); albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1: 268-276); alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985.Mot. Cell. Biol. 5: 1639-1648; and Hammer et al., 1987, Science 235:53-58); alpha 1-antitrypsin gene control region which is active in theliver (Kelsey et al., 1987, Genes and Devel. 1: 161-171); beta-globingene control region which is active in myeloid cells (Mogram et at.,1985, Nature 315: 338-340; and Kollias et al., 1986, Cell 46: 89-94);myelin basic protein gene control region which is active inoligodendrocyte cells in the brain (Readhead et at., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active inskeletal muscle (Sani, 1985, Nature 314: 283-286); swine alpha-skeletalactin control region which is active in muscle (Reecy, M. et al., 1998,Animal Biotechnology 9: 101-120) ; and gonadotropic releasing hormonegene control region which is active in the hypothalamus (Mason et al.,1986, Science 234: 1372-1378).

In a specific embodiment, a vector is used that comprises a promoteroperably linked to an antigenic peptide-encoding nucleic acid, one ormore origins of replication, and, optionally, one or more selectablemarkers (e.g., an antibiotic resistance gene). In another specificembodiment, a vector is used that comprises a promoter operably linkedto an antigenic fusion protein-encoding nucleic acid, one or moreorigins of replication, and, optionally, one or more selectable markers(e.g., an antibiotic resistance gene).

Expression vectors containing gene inserts can be identified by threegeneral approaches: (a) nucleic acid hybridization; (b) presence orabsence of “marker” gene functions; and (c) expression of insertedsequences. In the first approach, the presence of antigenicpeptide-encoding polynucleotides or antigenic fusion protein-encodingpolynucleotides inserted in an expression vector(s) can be detected bynucleic acid hybridization using probes comprising sequences that arehomologous to the inserted polynucleotide sequence. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of the gene(s) in the vector(s). For example, ifa nucleic acid molecule encoding an antigenic peptide or an antigenicfusion protein is inserted within the marker gene sequence of thevector, recombinants containing the nucleic acid molecule encoding theantigenic peptide or the antigenic fusion protein insert can beidentified by the absence of the marker gene function. In the thirdapproach, recombinant expression vectors can be identified by assayingthe gene product expressed by the recombinant. Such assays can be based,for example, on the physical or functional properties of an antigenicpeptide or an antigenic fusion protein in in vitro assay systems, e.g.,binding of an antigenic peptide or an antigenic fusion protein with ananti-IgE antibody.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g.,lambda), and plasmid and cosmid DNA vectors, to name but a few.

The term “host cell” as used herein refers not only to the particularsubject cell into which a recombinant DNA molecule is introduced butalso to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell strain may be chosen which modulates the expression of theinserted sequences, or modifies and processes the gene product in thespecific fashion desired. Expression from certain promoters can beelevated in the presence of certain inducers; thus, expression of thegenetically engineered may be controlled. Furthermore, different hostcells have characteristic and specific mechanisms for the translationaland post-translational processing and modification (e.g., glycosylation,phosphorylation of proteins). Appropriate cell lines or host systems canbe chosen to ensure the desired modification and processing of theforeign protein expressed. For example, expression in a bacterial systemcan be used to produce an unglycosylated core protein product.Expression in yeast will produce a glycosylated product. Expression inmammalian cells can be used to ensure “native” glycosylation of anantigenic peptide or antigenic fusion protein of the invention.Furthermore, different vector/host expression systems may effectprocessing reactions to different extents.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express anantigenic peptide or an antigenic fusion protein of the invention may beengineered. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA; engineered cells may be allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which in turn can be cloned andexpanded into cell lines. This method may advantageously be used toengineer cell lines which express an antigenic peptide or an antigenicprotein of the invention. Such engineered cell lines may be particularlyuseful in the screening and evaluation of anti-IgE antibodies or otheragents (e.g., organic molecules, inorganic molecules, organic/inorganiccomplexes, polypeptides, peptides, peptide mimics, polysaccharides,saccharides, glycoproteins, nucleic acids, DNA and RNA strands andoligonucleotides, etc.) that affect binding of an IgE molecule to itsreceptor.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11;223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ ceils, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981, Proc. Natl.Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolicacid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); neo,which confers resistance to the aminoglycoside G-418(Colberre-Garapin etal., 1981, J. Mol. Biol. 150: 1); and hygro, which confers resistance tohygromycin (Santerre et al., 1984, Gene 30: 147) genes.

In a specific embodiment, one or more nucleic acid molecules comprisinga polynucleotide sequence encoding an antigenic peptide of theinvention, are administered to treat or prevent IgE-mediated allergicdisorders, by way of gene therapy. In another specific embodiment, oneor more nucleic acid molecules comprising a polynucleotide sequenceencoding an antigenic fusion protein, are administered to treat orprevent IgE-mediated allergic disorders, by way of gene therapy. In yetanother specific embodiment, one or more nucleic acid moleculescomprising a polynucleotide sequence encoding an antigenic peptide ofthe invention, and one or more nucleic acid molecules comprising apolynucleotide sequence encoding an antigenic fusion protein of theinvention are administered to treat or prevent IgE-mediated allergicdisorders, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid, in this embodiment of the invention, thenucleic acids produce their encoded antigenic peptides or antigenicfusion proteins that mediate a therapeutic effect by eliciting an immuneresponse such as the production of anti-IgE antibodies.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993. Clinical Pharmacy 12: 488-505; Wu and Wu. 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 573-596;Mulligan, 1993, Science 260: 926-932; and Morgan and Anderson, 1993,Ann. Rev. Biochem. 62: 191-217; May, 1993, TIBTECH 11(5):155-215).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler,1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,NY.

In a preferred aspect, a pharmaceutical composition comprises nucleicacid sequences encoding an antigenic peptide of the invention, saidnucleic acid sequences being part of expression vectors that express theantigenic peptide in a suitable host. In particular, such nucleic acidsequences have promoters operably linked to the antigenic peptide codingregions, said promoters being inducible or constitutive, and,optionally, tissue-specific. In another preferred aspect, apharmaceutical composition comprises nucleic acid sequences encoding anantigenic fusion protein of the invention, said nucleic acid sequencesbeing part of expression vectors that express the antigenic fusionprotein in a suitable host. In particular, such nucleic acid sequenceshave promoters operably linked to the antigenic fusion protein codingregions, said promoters being inducible or constitutive, and,optionally, tissue-specific. In another particular embodiment, nucleicacid molecules are used in which the coding sequences of an antigenicpeptide of the invention and any other desired sequences are flanked byregions that promote homologous recombination at a desired site in thegenome, thus providing for intrachromosomal expression of the nucleicacids encoding the antigenic peptide (Koller and Smithies, 1989, Proc.Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al. 1989, Nature342:435-438). In another particular embodiment, nucleic acid moleculesare used in which the coding sequences of an antigenic fusion protein ofthe invention and any other desired sequences are flanked by regionsthat promote homologous recombination at a desired site in the genome,thus providing for intrachromosomal expression of the nucleic acidsencoding the antigenic protein.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992(Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et at.); WO92/20316dated Nov. 26, 1992 (Findeis et al); WO03/14188 dated Jul. 22, 1993(Clarke et al.); and WO 93/20221 dated Oct. 14,1993 (Young)).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86;8932-8935; Zijistra et al., 1989, Nature 342; 435-438).

In specific embodiments, viral vectors that contain nucleic acidsequences encoding antigenic peptides or antigenic fusion proteins areused. For example, a retroviral vector containing nucleic acid sequencesencoding an antigenic peptide or an antigenic fusion protein can be used(see, e.g., Miller et al., 1993, Meth. Enzymol. 217: 581-599). Theseretroviral vectors have been to delete retroviral sequences that are notnecessary for packaging of the viral genome and integration into hostcell DNA. The nucleic acid sequences encoding antigenic peptides orantigenic fusion proteins to be used in gene therapy are cloned into oneor more vectors, which facilitates delivery of the gene into a patient.More detail about retroviral vectors can be found in Boesen et al.,1994, Biotherapy 6: 291-302, which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., 1994, J. Clin. Invest 93: 644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129-141;and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3: 499-503present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5: 3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252: 431-434; Rosenfeld et al., 1992, Cell 68: 143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91: 225-234; PCT publicationWO94/12649; and Wang, et al., 1995, Gene Therapy 2: 775-783. In apreferred embodiment, adenovirus vectors are used. Adeno-associatedvirus (AAV) has also been proposed for use in gene therapy (see, e.g,Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204: 289-300; and U.S.Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a nucleic acidmolecule to cells in tissue culture by such methods as electroporation,lipofection, calcium phosphate mediated transfection, or viralinfection. Usually, the method of transfer includes the transfer of aselectable marker to the cells. The cells are then placed underselection to isolate those cells that have taken up and are expressingthe transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid molecule is introduced into a cellprior to administration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign nucleic acid molecules into cells (see, e.g., Loeffler and Behr,1993, Meth. Enzymol. 217: 599-618; Cohen el al., 1993, Meth. Enzymol.217: 618-644; Cline, 1985, Pharmac. Ther. 29: 69-92) and may be used inaccordance with the present invention, provided that the necessarydevelopmental and physiological functions of the recipient cells are notdisrupted. The technique should provide for the stable transfer of thenucleic acid to the cell, so that the nucleic acid is expressible by thecell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,subject's state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the subject.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding the antigenic peptides or antigenicfusion proteins of the invention are introduced into the cells such thatthey are expressible by the cells or their progeny, and the recombinantcells are then administered in vivo for therapeutic effect. In aspecific embodiment, stem or progenitor cells are used. Any stem and/orprogenitor cells which can be isolated and maintained in vitro canpotentially be used in accordance with this embodiment of the presentinvention (see e.g., PCT Publication WO 94/06598, dated Apr. 28, 1994;Stemple and Anderson, 1992, Cell 71: 973-985; Rheinwald, 1980, Meth.Cell Bio. 21A: 229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771).

in a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked lo thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

The invention also relates to methods for producing an antigenic peptideof the invention or an antigenic fusion protein of the inventioncomprising growing a culture of the cells of the invention in a suitableculture medium, and purifying the protein from the culture. For example,the methods of the invention include a process for producing anantigenic peptide or an antigenic fusion protein of the invention inwhich a host cell (i.e., a prokaryotic or eukaryotic celt) containing asuitable expression vector that includes a polynucleotide encoding anantigenic peptide or an antigenic fusion protein is cultured underconditions that allow expression of the encoded antigenic peptide or theencoded antigenic fusion protein. The antigenic peptide or the antigenicfusion protein can be recovered from the culture, conveniently from theculture medium, and further purified. The purified antigenic peptides orantigenic fusion proteins can be used in in vitro immunoassays which arewell known in the art to identify anti-IgE antibodies which bind to theantigenic peptides or the antigenic fusion proteins.

The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MaxBat.RTM. kit), and such methods arewell known in the art, as described in Summers and Smith, TexasAgricultural Experiment Station Bulletin No. 1555 (1987), incorporatedherein by reference. As used herein, an insect cell capable ofexpressing a polynucleotide of the present invention is “transformed.”

Alternatively, an antigenic peptide of the Invention or an antigenicfusion protein of the invention may also be expressed in a form whichwill facilitate purification. For example, an antigenic peptide may beexpressed as fusion protein comprising a heterologous protein such asmaltose binding protein (MBP) glutathione-S-transferase (GST) orthioredoxin (TRX) which facilitate purification. Kits for expression andpurification of such fusion proteins are commercially available from NewEngland BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogen, respectively. The protein can also be tagged with an epitopeand subsequently purified by using a specific antibody directed to suchepitope. One such epitope (“Flag”) is commercially available from Kodak(New Haven, Conn.).

The antigenic peptides of the invention or the antigenic fusion proteinsof the invention may also be expressed as a product of transgenicanimals, e.g., as a component of the milk of transgenic cows, goats,pigs, or sheep which are characterized by somatic or germ cellscontaining a nucleotide sequence encoding the antigenic peptide or theantigenic fusion protein.

Any method known to those of skill in the art can be used to produce anantigenic peptide or an antigenic fusion protein of the invention. Atthe simplest level, the amino acid sequence can be synthesized usingcommercially available peptide synthesizers. This is particularly usefulin producing small peptides and fragments of larger polypeptides. Theisolated antigenic peptides and antigenic fusion proteins of theinvention are useful, for example, in generating antibodies against thenative polypeptide.

One skilled in the art can readily follow known methods for isolatingpeptides and proteins in order to obtain one of the isolated antigenicpeptides or antigenic fusion proteins of the present invention. Theseinclude, but are not limited to, immunochromatography, high performanceliquid chromatography (HPLC), reverse-phase high performance liquidchromatography (RP-HPLC), size-exclusion chromatography, ion-exchangechromatography, and immuno-affinity chromatography. See, e.g., Scopes,Protein Purification: Principles and Practice, Springer-Verlag (1994);Sambrook et al., in Molecular Cloning: A Laboratory Manual; Ausubel etal., Current Protocols in Molecular Biology.

An antigenic peptide or an antigenic fusion protein of the invention is“isolated” or “purified” when it is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the protein is derived, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinant produced. Thus,protein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, or 5% (bydry weight) of a contaminating protein. When an antigenic peptide or anantigenic fusion protein of the invention is recombinantly produced, itis also preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, 10%, or 5% of the volume of theprotein preparation. When an antigenic peptide or an antigenic fusionprotein of the invention is produced by chemical synthesis, it ispreferably substantially free of chemical precursors or other chemicals,i.e., it is separated from chemical precursors or other chemicals whichare involved in the synthesis of the antigenic peptide or the antigenicfusion protein. Accordingly such preparations of the protein have lessthan about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors orcompounds other than the antigenic peptide or the antigenic fusionprotein.

The compositions of the invention are preferably tested in vitro, andthen in vivo for the desired therapeutic or prophylactic activity, priorto use in humans. For example, in vitro assays which can be used todetermine whether administration of a specific composition is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered acomposition, and the effect of such composition upon the tissue sampleis observed.

The expression of an antigenic peptide or an antigenic fusion proteincan be assayed by the immunoassays, gel electrophoresis followed byvisualization, or any other method known to those skilled in the art.

In various specific embodiments, in vitro assays can be carried out withrepresentative cells of cell types involved in a patient's disorder, todetermine if a composition has a desired effect upon such celt types. Inaccordance with the present invention, the functional activity of anantigenic peptide or an antigenic fusion protein can be measured by itsability to induce anti-IgE antibodies that inhibit IgE from binding toits receptor on mast cells or basophils in vitro without inducing therelease of vasoactive substances such as histamine.

Compositions for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to pigs,chicken, cows or monkeys.

The invention provides methods of treatment (and prophylaxis) byadministration to a subject of an effective amount of a composition ofthe invention to elicit the production of anti-IgE antibodies which donot cause anaphylaxis. In a preferred aspect, a composition of theinvention is substantially purified (e.g., substantially free fromsubstances that limit its effect or produce undesired side-effects). Thesubject is preferably an animal, including but not limited to animalssuch as cows, pigs, horses, chickens, cats, dogs, etc., and ispreferably a mammal, and most preferably human.

Formulations and methods of administration that can be employed when thecomposition comprises a nucleic acid are described above; additionalappropriate formulations and routes of administration can be selectedfrom among those described herein below.

Various delivery systems are known and can be used to administer acomposition of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe composition, receptor-mediated endocytosis (see, e.g., Wu and Wu,1987. J. Biol. Chem. 262: 4429-4432), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intratumoral, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compositions may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, pulmonary administration can be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, topical application, injection, or by meansof an implant, said implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.In one embodiment, administration can be by direct injection at the site(or former site) of an allergic reaction.

In another embodiment, a composition of the invention can be deliveredin a vesicle, in particular a liposome (see, e.g., Langer, 1990, Science249: 1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); and Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

In yet another embodiment, a composition of the invention can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see, e.g., Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed.Eng. 14: 201; Buchwald et al., 1980, Surgery 88: 507; and Saudek et al.,1989, N. Engl. J. Med. 321: 574). In another embodiment, polymericmaterials can be used (see Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also Levy et al., 1985,Science 228: 190; During et al., 1989, Ann. Neurol. 25: 351; and Howardet at.,1989, J. Neurosurg. 71: 105). In yet another embodiment, acontrolled release system can be placed in proximity of the therapeutictarget, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(1990, Science 249: 1527-1533).

In a specific embodiment where the composition of the invention is anucleic acid encoding an antigenic peptide or an antigenic fusionprotein of the invention, the nucleic acid can be administered in vivoto promote expression of its encoded antigenic peptide or antigenicfusion protein, by constructing it as part of an appropriate nucleicacid expression vector and administering it so that it becomesintracellular, e.g., by use of a retroviral vector (see U.S. Pat. No.4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic. Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-iike peptide which is known toenter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci.USA 88: 1864-1868), etc. Alternatively, a nucleic acid can be introducedintracellular and incorporated within host cell DNA for expression, byhomologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of an antigenicpeptide or an antigenic fusion protein of the invention, and apharmaceutical acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,excipient, or vehicle with which the therapeutic is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water is a preferred carrier when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. The composition, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of theantigenic peptide or the antigenic fusion protein, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The antigenic peptides or antigenic fusion proteins of the invention canbe formulated as neutral or salt forms. Pharmaceutically acceptablesalts include those formed with free amino groups such as those derivedfrom hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with free carboxyl groups such as those derived fromsodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine,triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The following examples further illustrate the invention.

EXAMPLES

1. Selection of antigenic peptides and cloning of correspondingpolyneucleotide sequences

A major obstacle facing the development of an anti-IgE vaccine is thelack of information regarding the precise amino acids representingnon-anaphylactogenic IgE determinants that could be safely used toimmunize allergic subjects and induce non-anaphylactogenic polyclonalantibodies (i.e. polyclonal anti-IgE antibodies that do not bind toreceptor-bound IgE). By definition, those determinants ideallycorrespond to only those IgE amino acid sequences that physicallycontact the IgE receptor. Clearly, there is no information in the priorart on the precise identity of those sequences. Indeed, the prior art isinconsistent on even the region or domain of IgE within which thosesequences may reside. Furthermore, the identity of non-anaphylactogenicdeterminants could be correctly elucidated from only solving the crystalstructure of IgE-IgE receptor complex which, unfortunately, has not yetbeen achieved. Consequently, the present invention overcome thisobstacle and provide IgE determinants capable of inducing withinallergic hosts therapeutically desirable polyclonal antibodies thatreact with native serum IgE, prevent IgE from binding to its receptor onmast cells and basophils and do not react with receptor-bound IgE. Inorder to identify non-anaphylactogenic IgE epitopes suitable forinclusion into an anti-IgE vaccine, we follow an approach that does notmake any a priori assumptions about the location or require knowledge ofthe exact amino acids on IgE suitable for an effective and safe vaccine.The IgE antibody has been to shown to exist in many species throughoutthe animal kingdom including for example humans, dogs, cats, sheep,cows, pigs, horses, rats, mice and chimpanzee. Indeed the IgE gene andits encoded protein have been identified in ail these species.Comparison of the primary amino acid sequence among these IgE moleculesshows that they share common amino acid sequences in many locationsthroughout the IgE molecule. These common (conserved) sequences areflanked by amino acid sequences that vary among the various IgEmolecules. We reasoned that a comparison of the primary sequence of IgEmolecules from different species e.g. rat IgE and dog IgE would provideclues to identification of non-anaphylactogenic IgE determinants. It isknown that dog, cat, and rat IgE bind to the human IgE receptor. SinceIgE from dog and rat bind to the IgE receptor of another unrelatedspecies such as human receptor, we hypothesize that the conserved aminoacids between rat and dog must contain the information specifying thereceptor-binding conformational determinants. Since these conservedsequences are flanked within their respective IgE molecule withsequences that vary between dog and rat IgE, we further hypothesize thatthe variable IgE sequences could be exchanged between IgE from dog andrat without affecting the overall receptor-binding conformation ofeither IgE molecules. Using this reasoning, a safe and effective vaccinefor dogs could be developed by using peptides of the present inventionSEQ ID NOS: 1 to 6. The nucleotide sequences encoding antigenic peptidesof the present invention were prepared using the following procedures:

Cloning of dog CH3 domain (SEQ ID NO: 15).

The polyneucteotide sequence encoding dog CH3 domain was assembled by apolymerase chain reaction (PCR)-based gene synthesis procedure. A set ofoligonucleotide primers (listed in Table 1) was synthesized at LifeTechnologies Inc.

TABLE 1 Primers for cloning of Dog CH3 domain DNA (SEQ ID NO: 15) PrimerPrimer sequence name AAGCGTGCCCCCCCGGAAGTCTATGCGTTTGCGAC P173-S712TCGGGGGTCGGACTCTGAACACTTCTTGGTGCTGTC P173-A402GACAGCACCAAGAAGTGTTCAGAGTCCGACCCCCGAGGCG P173-S1 TGACGAGCTACCTGAGCCCACCCAGCCCCCTTGACCTGTATGTC P173-S2CACAAGGCGCCCAAGATCACCTGCCTGGTAGTGGACCTGG P173-S3CCACCATGGAAGGCATGAACCTGACCTGGTACCGGGAGAG P173-S4CAAAGAACCCGTGAACCCGGGCCCTTTGAACAAGAAGGAT P173-S5CACTTCAATGGGACGATCACAGTCACGTCTACCCTGCCAG P173-S6TGAACACCAATGACTGGATCGAGGGCGAGACCTACTATTG P173-S7CAGGGTGACCCACCCGCACCTGCCCAAGGACATCGTGCGC P173-S8TCCATTGCCAAGGCCCCTGGCAAGCGTGCCCCCCCGGAAG P173-S9CGGCGTCGCAAACGCATAGACTTCCGGGGGGGCACGCTTG P173-A1CCAGGGGCCTTGGCAATGGAGCGCACGATGTCCTTGGGCA P173-A2GGTGCGGGTGGGTCACCCTGCAATAGTAGGTCTCGCCCTC P173-A3GATCCAGTCATTGGTGTTCACTGGCAGGGTAGACGTGACT P173-A4GTGATCGTCCCATTGAAGTGATCCTTCTTGTTCAAAGGGC P173-A5CCGGGTTCACGGGTTCTTTGCTCTCCCGGTACCAGGTCAG P173-A6GTTCATGCCTTCCATGGTGGCCAGGTCCACTACCAGGCAG P173-A7GTGATCTTGGGCGCCTTGTGGACATACAGGTCAAGGGGGC P173-A8TGGGTGGGCTCAGGTAGCTCGTCACGCCTCGGGGGTCGGA P173-A9

These primers were used to assemble the dog CH3 domain in a two-step PCRreaction as follows: 1) 25 cycles using an equimolar mixture of 18primers (P173-S1 to -S9 and P173-A1 to -A9) followed by 2) dilution ofthe product from step 1 (0.625 ul into a 50 ul reaction) into a newreaction and carrying out 15 cycles of PCR using the two terminalprimers (P173-S1 and P173-A1). All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. ThisPCR reaction resulted in amplification of a gene sequence of the correctsize.

Cloning of nucleotide sequence encoding partial Human CH3/partial dogCH3 domain fusion protein (SEQ ID NO: 16).

This DNA sequence encodes a protein which consists of the first 63 aminoacids of human CH3 domain fused to the last 53 amino acid of the dog CH3domain. The polynucleotide sequence encoding this construct wasassembled as follows: A set of oligonucleotide primers (listed in table2) was synthesized at Life Technologies Inc.

TABLE 2 Primers for cloning of human/dog CH3 domain fusion DNA (SEQ IDNO: 16) Primer Primer sequence nameCCTCTCGGGTTGGAATCTGCACACTTCTTGGTGCTGTC P174-A404AAGCGTGCCCCCCCGGAAGTCTATGCGTTTGCGAC P174-S721GACAGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGT P174-S1 GAGCGCCTACCTAAGCCGGCCCAGCCCGTTCGACCTGTTCATC P174-S2CGCAAGTCGCCCACGATCACCTGTCTGGTGGTGGACCTGG P174-S3CACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGC P174-S4CAGTGGGAAGCCTGTGAACCACTCCACCAGAAGGAGGAG P174-S5AAGAAGGATCACTTCAATGGGACGATCACAGTCACGTCTA P174-S6CCCTGCCAGTGAACACCAATGACTGGATCGAGGGCGAGAC P174-S7CTACTATTGCAGGGTGACCCACCCGCACCTGCCCAAGGAC P174-S8ATCGTGCGCTCCATTGCCAAGGCCCCTGGCAAGCGTGCCC P174-S9AAACGCATAGACTTCCGGGGGGGCACGCTTGCCAGGGGCC P174-A1TTGGCAATGGAGCGCACGATGTCCTTGGGCAGGTGCGGGT P174-A2GGGTCACCCTGCAATAGTAGGTCTCGCCCTCGATCCAGTC P174-A3ATTGGTGTTCACTGGCAGGGTAGACGTGACTGTGATCGTC P174-A4CCATTGAAGTGATCCTTCTTCTCCTCCTTTCTGGTGGAGT P174-A5GGTTCACAGGCTTCCCACTGGCCCGGGACCAGGTCAGGTT P174-A6CACGGTCCCCTTGCTGGGTGCCAGGTCCACCACCAGACAG P174-A7GTGATCGTGGGCGACTTGCGGATGAACAGGTCGAACGGGC P174-A8TGGGCCGGCTTAGGTAGGCGCTCACCCCTCTCGGGTTGGA P174-A9

These primers were used to assemble the human CH3/dog CH3 domain fusionin a two-step PCR reaction as follows: 1) 25 cycles using an equimolarmixture of 18 primers (P174-S1 to -S9 and P174-A1 to -A9) followed by 2)dilution of the product from step 1 (0.625 μl into a 50 μl reaction)into a new reaction and carrying out 15 cycles of PCR using the twoterminal primers (P174-S1 and P174-A1). All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. ThisPCR reaction resulted in amplification of a gene sequence of the correctsize (384 nucleotides).

Cloning of chimeric Human/dog CH3 domain (SEQ ID NO: 17).

This DNA sequence encodes a protein, which consists of alternatinghuman/dog CH3domain sequences. The polyneucleotide sequence encodinghuman CH3/conserved dog CH3 domain was assembled as follows: A set ofoligonucleotide primers (listed in Table 3) was synthesized at LifeTechnologies Inc.

TABLE 3 Primers for cloning of human/dog CH3 domain chimeric DNA (SEQ IDNO: 17) Primer Primer Sequence nameGACAGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGT P175-S1 GACCAGCTACCTAAGCCCGCCCAGCCCGCTGGACCTGTACATC P175-S2CGCAAGTCGCCCAAGATCACCTGTCTGGTGGTGGACCTGG P175-S3CACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGC P175-S4CAGTGGGAAGCCTGTGAACCACTCCACCAGAAAGGAGGAG P175-S5AAGCAACGGAATGGGACGATCACAGTCACGTCTACCCTGC P175-S6CAGTGGGCACCAGAGACTGGATCGAGGGCGAGACCTACTA P175-S7TTGCAGGGTGACCCACCCGCACCTGCCCAAGGACATCGTG P175-S8CGCTCCATTGCCAAGGCCCCTGGCAAGCGTGCCCCCCCGG P175-S9CGTCGCAAACGCATAGACTTCCGGGGGGGCACGCTTGCCA P175-A1GGGGCCTTGGCAATGGAGCGCACGATGTCCTTGGGCAGGT P175-A2GCGGGTGGGTCACCCTGCAATAGTAGGTCTCGCCCTCGAT P175-A3CCAGTCTCTGGTGCCCACTGGCAGGGTAGACGTGACTGTG P175-A4ATCGTCCCATTCCGTTGCTTCTCCTCCTTTCTGGTGGAGT P175-A5GGTTCACAGGCTTCCCACTGGCCCGGGACCAGGTCAGGTT P175-A6CACGGTCCCCTTGCTGGGTGCCAGGTCCACCACCAGACAG P175-A7GTGATCTTGGGCGACTTGCGGATGTACAGGTCCAGCGGGC P175-A8TGGGCGGGCTTAGGTAGCTGGTCACCCCTCTCGGGTTGGA P175-A9CCTCTCGGGTTGGAATCTGCACACTTCTTGGTGCTGTCCT P175-A404AAGCGTGCCCCCCCGGAAGTCTATGCGTTTG P175-S715

These primers were used to assemble the human CH3/dog CH3 chimericdomain in a two-step PCR reaction as follows: 1) 25 cycles using anequimolar mixture of 18 primers (P175-S1 to -S9 and P175-A1 to -A9)followed by 2) dilution of the product from step 1 (0.625 ul into a 50ul reaction) into a new reaction and carrying out 15 cycles of PCR usingthe two terminal primers (P175-S1 and P175-A1). All reactions used BMBExpand HE polymerase mixture and conditions specified by themanufacturer. This PCR reaction resulted in amplification of a genesequence of the correct size (384 nucleotides).

Cloning of human CH3 domain (SEQ ID NO: 18).

The polyneucleotide sequence encoding human CH3 domain was assembled asfollows: A set of oligonucleotide primers (listed in Table 4) wassynthesized at Life Technologies Inc.

TABLE 4 Primers for Human CH3 domain DNA (SEQ ID NO: 18) Primer Primersequence name GACAGCACCAAGAAGTGTGCAGATTCCAACCCGAGAGGGGT P176-S1 GAGCGCCTACCTAAGCCGGCCCAGCCCGTTCGACCTGTTCATC P176-S2CGAAGTCGCCCACGATCACCTGTCTGGTGGTGGACCTGG P176-S3CACCCAGCAAGGGGACCGTGAACCTGACCTGGTCCCGGGC P176-S4CAGTGGGAAGCCTGTGAACCACTCCACCAGAAAGGAGGAG P176-S5AAGCAGCGCAATGGCACGTTAACCGTCACGTCCACCCTGC P176-S6CGGTGGGCACCCGAGACTGGATCGAGGGGGAGACCTACCA P176-S7GTGCAGGGTGACCCACCCCCACCTGCCCAGGGCCCTCATG P176-S8CGGTCCACGACCAAGACCAGCGGCCCGCGTGCTGCCCCGG P176-S9CGTCGCAAACGCATAGACTTCCGGGGCAGCACGCGGGCCG P176-A1CTGGTCTTGGTCGTGGACCGCATGAGGGCCCTGGGCAGGT P176-A2GGGGGTGGGTCACCCTGCACTGGTAGGTCTCCCCCTCGAT P176-A3CCAGTCTCGGGTGCCCACCGGCAGGGTGGACGTGACGGTT P176-A4AACGTGCCATTGCGCTGCTTCTCCTCCTTTCTGGTGGAGT P176-A5GGTTCACAGGCTTCCCACTGGCCCGGGACCAGGTCAGGTT P176-A6CACGGTCCCCTTGCTGGGTGCCAGGTCCACCACCAGACAG P176-A7GTGATCGTGGGCGACTTGCGGATGAACAGGTCGAACGGGC P176-A8TGGGCCGGCTTAGGTAGGCGCTCACCGCTCTCGGGTTGGA P176-A9CCTCTCGGGTTGGAATCTGCACACTTCTTGGTGCT P176-A404GCGGCCCGCGTGCTGCCCCGGAAGTCTATGCGTTTGCGAC P176-S710

These primers were used to assemble the human CH3 domain in a two-stepPCR reaction as follows: 1) 25 cycles using an equimolar mixture of 18primers (P176-S1 to S9and P176-A1 to -A9) followed by 2) dilution of theproduct from step 1 (0.625 ul into a 50 ul reaction) into a new reactionand carrying out 15 cycles of PCR using the two terminal primers(P176-S1 and P176-A1). All reactions used BMB Expand HF polymerasemixture and conditions specified by the manufacturer. This PCR reactionresulted in amplification of a gene sequence of the correct size (384nucleotides).

Cloning of rat CH3 domain (SEQ ID NO: 19).

The polyneucleotide sequence encoding rat CH3 domain was assembled asfollows: A set of oligonucleotide primers (listed in Table 5) wassynthesized at Life Technologies Inc.

TABLE 5 Primers for Rat CH3 DNA (SEQ ID NO: 19) Primer Primer sequencename GACAGCACCAAGAAGTGCTCAGATGATGAGCCCCGGGGTGT P177-S1 GATTACCTACCTGATCCCACCCAGTCCCCTCGACCTGTATGAA P177-S2AATGGGACTCCCAAACTTACCTGTCTGGTTTTGGACCTGG P177-S3AAAGTGAGGAGAATATCACCGTGACGTGGGTCCGAGAGCG P177-S4TAAGAAGTCTATAGGTTCGGCATCCCAGAGGAGTACCAAG P177-S5CACCATAATGCCACAACCAGTATCACCTCCATCTTGCCAG P177-S6TGGATGCCAAGGACTGGATCGAAGGTGAAGGCTACCAGTG P177-S7CAGAGTGGACCACCCTCACTTTCCCAAGCCCATTGTGCGT P177-S8TCCATCACCAAGGCCCCAGGCAAGCGCTCAGCCCCAGAAG P177-S9CGGCGTCGCAAACGCATAGACTTCTGGGGCTGAGCGCTTG P177-A1CCTGGGGCCTTGGTGATGGAACGCACAATGGGCTTGGGAA P177-A2AGTGAGGGTGGTCCACTCTGCACTGGTAGCCTTCACCTTC P177-A3GATCCAGTCCTTGGCATCCACTGGCAAGATGGAGGTGATA P177-A4CTGGTTGTGGCATTATGGTGCTTGGTACTCCTCTGGGATG P177-A5CCGAACCTATAGACTTCTTACGCTCTCGGACCCACGTCAC P177-A6GGTGATATTCTCCTCACTTTCCAGGTCCAAAACCAGACAG P177-A7GTAAGTTTGGGAGTCCCATTTTCATACAGGTCGAGGGGAC P177-A8TGGGTGGGATCAGGTAGGTAATCACACCCCGGGGCTCATC P177-A9CGGGGCTCATCATCTGAGCACTTCTTGGTGCTGTCCT P177-A401CAAGCGCTCAGCCCCAGAAGTCTATGCGTTTGCGAC P177-S711

These primers were used to assemble the rat CH3 domain in a two-step PCRreaction as follows: 1) 25 cycles using an equimolar mixture of 18primers (P177-S1 to -S9and P177-A1 to -A9) followed by 2) dilution ofthe product from step 1 (0.625 μl into a 50 μl reaction) into a newreaction and carrying out 15 cycles of PCR using the two terminalprimers (P177-S1 and P177-A1). All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. ThisPCR reaction resulted in amplification of a gene sequence of the correctsize (384 nucleotides).

Cloning of human CH3 for expression in baculovirus (SEQ ID NO: 20).

The polyneucleotide sequence encoding the IgE CH3 domain and the signalsequence from honey-bee mellitin was assembled as follows: A set ofoligonucleotide primers (listed in Table 6) was synthesized at LifeTechnologies Inc.

TABLE 6 Primers for baculovirus expressed Human IgE CH3 domain (SEQ IDNO: 20) Primer Primer sequence nameGCGGATCCATGAAATTCTTAGTCAACGTTGCCCTTGTTTTAT P158-S1GGTCGTATACATTTCTTACATCTATGCGGACAGCAACCCG P158-S2AGAGGGGTGAGCGCCTACCTAAGCCGGCCCAGCCCGTTCG P158-S3ACCTGTTCATCCGCAAGTCGCCCACGATCACCTGTCTGGT P158-S4GGTGGACCTGGCACCCAGCAAGGGGACCGTGAACCTGACC P158-S5TGGTCCCGGGCCAGTGGGAAGCCTGTGAACCACTCCACCA P158-S6GAAAGGAGGAGAAGCAGCGCAATGGCACGTTAACCGTCAC P158-S7GTCCACCCTGCCGGTGGGCACCCGAGACTGGATCGAGGGG P158-S8GAGACCTACCAGTGCAGGGTGACCCACCCCCACCTGCCCA P158-S9GGGCCCTCATGCGGTCCACGACCAAGACCTCCTGATGAATTC P158-S10 CCG P158-A1CCGGAATTCATCAGGAGGTCTTTGGT P158-A2CGTGGACCGCATGAGGGCCCTGGGCAGGTGGGGGTGGGTC P158-A3ACCCTGCACTGGTAGGTCTCCCCCTCGATCCAGTCTCGGG P158-A4TGCCCACCGGCAGGGTGGACGTGACGGTTAACGTGCCATT P158-A5GCGCTGCTTCTCCTCCTTTCTGGTGGAGTGGTTCACAGGC P158-A6TTCCCACTGGCCCGGGACCAGGTCAGGTTCACGGTCCCCT P158-A7TGCTGGGTGCCAGGTCCACCACCAGACAGGTGATCGTGGG P158-A8CGACTTGCGGATGAACAGGTCGAACGGGCTGGGCCGGCTT P158-A9AGGTAGGCGCTCACCCCTCTCGGGTTGCTGTCCGCATAGA P158-S10TGTAAGAAATGTATACGACCATAAAAACAAGGGCAACGTT

These primers were used to assemble the human CH3 domain in a two-stepPCR reaction as follows: 1) 25 cycles using an equimolar mixture of 20primers (P158-S1 to -S10 and P158-A1 to -A10) followed by 2) dilution ofthe product from step 1 (1.25 ul into a 50 ul reaction) into a newreaction and carrying out 10 cycles of PCR using primers (P158-S1 andP158 A1). All reactions used BMB Expand HF polymerase mixture andconditions specified by the manufacturer. This PCR reaction resulted inamplification of a gene sequence of the correct size (400 nucleotides).The PCR amplified 409 bp fragment was digested with EcoRI and BamHIenzymes and ligated to pFastBac1 plasmids digested with EcoRI and BamHIenzymes. The ligation mixture was transformed into DH5 E. coli andcolonies containing the plasmid plus 409 bp insert were isolated.Plasmid DNA was prepared from DHS cells using Quiagen columns accordingto the manufacturer's recommendation.

The “donor plasmid” (pFastBac1-CH3) DNA was transformed into DH10Baccompetent cells for transposition into the bacmid according to theBac-To-Bac Baculovirus Expression System protocol (Life Technologies).White colonies that contained the recombinant bacmid were isolated andgrown up for isolation of bacmid DNA. To isolate bacmid DNA, ConcertHigh Purity Plasmid isolation System (Life Technologies) was usedaccording to the methods provided by the manufacturer. PCR analysis ofrecombinant bacmid was used to confirm that the CH3 gene had transposedinto the bacmid. PUC/M13 amplification primers (Life Technologies) wereused in reaction conditions specified by the Bac-To-Bac ExpressionSystems manual. The reaction yielded a single specific product 2709 basepairs in size indicating that the CH3 domain gene was inserted into thebacmid (bacmid transposed with pFastBac1 2300 bp+CH3 domain 409 bp=2709bp).

2. Selection of protein carrier and cloning of correspondingpolyneucleotide sequences.

The antigenic peptides of the present invention were incorporated withina carrier protein whose function is to increase the immunogenicity ofthe peptides and at the same time preserve the conformational attributesof these peptides that are necessary to induce the appropriate anti-IgEantibodies. For this purpose, a carrier system was developed based onthe utilization of a modified CH2 and CH4 domains of human IgE. Themodification of human CH2 and CH4 domain were introduced so as to avoidimmunological cross-reactivity between human CH2-CH4 protein sequenceand dog CH2-CH-4 protein sequence. The amino acid sequence of thecarrier protein SEQ ID MO: :7-9 was cloned using the followingprocedures:

Cloning of Human CH2 Domain (SEQ ID NO: 21):

The polynucleotide sequence encoding signal sequence::human CH2 domainwas assembled by a two step polymerase chain reaction (PCR)-based genesynthesis procedures follows: A set of oligonucleotide primers (listedin Table 7) was synthesized at Life Technologies Inc.

TABLE 7 Primers for Human CH2 domain DNA (SEQ ID NO: 21) Primer Primersequence name GACTGCTAGCCATGAGTGTGCCCA P171-S1bGACTGCTAGCCATGAGTGTGCCCACTCAGGTCCTGGGGTT P171-S1GCTGCTGCTGTGGCTTACAGATGCCAGATGTGACATCGTC P171-S2GCCTCCAGGGACTTCACCCCGCCCTCCGTGAAGATCTTAC P171-S3AGTCGTCCTGCGACGGCGGCGGGCACTTCCCCCCGACCAT P171-S4CCAGCTCTACTGCCTCGTCTCTGGGTACACCCCAGGGACT P171-S5ATCCAGATCACCTGGCTGGAGGACGGGCAGGTCATGGACG P171-S6TGGACTTGTCCACCGCCTCTAGCACGCAGGAGGGTGAGCT P171-S7GGCCTCCACACAAAGCGAGCTCACCCTCAGCCAGAAGCAC P171-S8TGGCTGTCAGACCGCACCTTCACCTGCCAGGTCACCTATC P171-S9AAGGTCACACCTTTGAGGACAGCACCAAGAAGTGTCTCGA P171-S10GACTCTCGAGACACTTCTTGGTGCT P171-A1GTCCTCAAAGGTGTGACCTTGATAGGTGACCTGGCAGGTG P171-A2AAGGTGCGGTCTGACAGCCAGTGCTTCTGGCTGAGGGTGA P171-A3GCTCGCTTTGTGTGGAGGCCAGCTCACCCTCCTGCGTGGT P171-A4AGAGGCGGTGGACAAGTCCACGTCCATGACCTGCCCGTCC P171-A5TCCAGCCAGGTGATCTGGATAGTCCCTGGGGTGTACCCAG P171-A6AGACGAGGCAGTAGAGCTGGATGGTCGGGGGGAAGTGCCC P171-A7GCCGCCGTCGCAGGACGACTGTAAGATCTTCACGGAGGGC P171-A8GGGGTGAAGTCCCTGGAGGCGACGATGTCACATCTGGCAT P171-A9CTGTAAGCCACAGCAGCAGCAACCCCAGGACCTGAGTGGG P171-A10 TGGCTGTCAGACCGCACCTTCAP171-S321 ACTTCTTGGTGCTGTCCTCA P171-A393

These primers were used to assemble the signal sequence::human CH2domain in a two-step PCR reaction as follows: 1) 25 cycles using anequimolar mixture of 20 primers (P171-S1 to -S10 and P171-A1 to -A10)followed by 2) dilution of the product from step 1 (0.625 ul into a 50ul reaction) into a new reaction and carrying out 15 cycles of PCR usingthe two terminal primers (P17-S1b and P171-A1). All reactions used BMBExpand HF polymerase mixture and conditions specified by themanufacturer. This PCR reaction resulted in amplification of a genesequence of the correct size. The amplified gene was then cloned into(pGEM-T) vector at the T/A cloning site. The nucleotide sequence of theamplified fragment was determined by automated fluorescent DNAsequencing.

Cloning of Human CH4 Domain (SEQID#22).

The polynucleotide sequence encoding human CH4 domain was assembled by atwo step polymerase chain reaction (PCR)-based gene synthesis procedureas follows: A set of oligonucleotide primers (listed in Table 8) wassynthesized at Life Technologies Inc.

TABLE 8 Primers for Human CH4 DNA (SEQ ID NO: 22) Primer Primer sequencename GACTCTCGAGGAAGTCTATGCGTT P172-S1bGACTCTCGAGGAAGTCTATGCGTTTGCGACGCCGGAGTGG P172-S1CCGGGGAGCCGGGACAAGCGCACCCTCGCCTGCGTGGTGC P172-S2AGAACTTCATGCCTGAGGACATCTCGGTGCGCTGGCTGCA P172-S3CAACGAGGTGCAGCTCCCGGACGCCCGGCACAGCACGACG P172-S4CAGCCCCGCAAGACCAAGGGCTCCGGCTTCTTCGTCTTCA P172-S5GCCGCCTGGCGGTGACCAGGGCCGAATGGCAGGAGAAAGA P172-S6TGAGTTCATCTGCCGTGCAGTCCATGAGGCAGCGAGCCCC P172-S7TCACAGACCGTCCAGCGAGCGGTGTCTGTAAATCCCGGTA P172-S8GACTGAATTCTCATTTACCGGGATT P172-A1b GACTGAATTCTCATTTACCGGGATTTACAGACACCP172-A1 GCTCGCTGGACGGTCTGTGAGGGGCTCGCTGCCTCATGGA P172-A2CTGCACGGCAGATGAACTCTTTCTCCTGCCATTCGGC P172-A3CCTGGTCACCGCCAGGCGGCTGAAGACGAAGAAGCCGGAG P172-A4CCCTTGGTCTTGCGGGGCTGCGTCGTGCTGTGCCGGGCGT P172-A5CCGGGAGCTGCACCTCGTTGTGCAGCCAGCGCACCGAGAT P172-A6GTCCTCAGGCATGAAGTTCTGCACCAGGCAGGCGAGGGTG P172-A7CGCTTGTCCCGGCTCCCCGGCCACTCCGGCGTCGCAAACG P172-A8 GAAGTCTATGCGTTTGCGACGP172-S11 GCAGCCAGCGCACCGAGATGTC P172-A119

These primers were used to assemble the human CH4 domain in a two stepPCR reaction as follows: 1) 25 cycles using an equimolar mixture of 16primers (P172-S1 to -S8 and P172-A1 to -A8) followed by 2) dilution ofthe product from step 1 (0.62 ul into a 50 ul reaction) into a newreaction and carrying out 15 cycles of PCR using the two terminalprimers (P172-S1b and P172-A1b). All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. ThisPCR reaction resulted in amplification of a gene sequence of the correctsize. The amplified gene was then cloned into (pGEM-T) vector at the T/Acloning site. The nucleotide sequence of the amplified fragment wasdetermined by automated fluorescent DNA sequencing.

3. Cloning of genes encoding fusion protein vaccines:

Polynucleotide sequences encoding fusion proteins for use as vaccines(SEQ ID NO: 24-28) of the present invention were prepared as follows.

Cloning of IgE-1 vaccine construct (SEQ ID NO: 24):

The insert in construct IgE-1 consists of the signal sequence-human CH2domain followed by the dog CH3 domain followed by the human CH4 domain.Assembly of the insert for IgE-1 consisted of using the signalsequence-human CH2 domain as a template for one PCR reaction, and thehuman CH4 domain as a template for a second PCR reaction. In these tworeactions, terminal primers were used to generate regions of homologywith the dog CH3 domain, so that the two ends of the dog CH3 domain DNAfragment would hybridize to the two human domain DNA fragments and thethree fragments would serve as “megaprimers” in a PCR reaction. The dogCH3 domain was engineered to contain overlapping sequence on either endto the two human domain fragments (CH2 homology on the 5′ end and CH4homology on the 3′ end). Then, the three PCR fragments (human CH2, dogCH3 and human CH4) were mixed in a final PCR reaction utilizing theterminal primers to generate a full-length product. The procedure isoutlined as follows: Fragment 1: (signal sequence-human CH2 domain) 413bp fragment resulting from amplification of human CH2domain with primersP171-S1b and P173-A402; fragment was amplified in 35 cycles of PCR,followed by gel purification. All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer.Fragment 2: (dog CH3 domain) 384 bp fragment described above; gelpurified Fragment 3: (human CH4 domain) 340 bp fragment resulting fromamplification of human CH4 domain with primers P173-S712 and P172-A1b;fragment was amplified in 35 cycles of PCR, followed by gelpurification. All reactions used BMB Expand HF polymerase mixture andconditions specified by the manufacturer. The three fragments were addedin approximately equimolar amounts to a final PCR reaction using the twoterminal primers P171-S1b and P172-A1b, and carrying out 35 cycles ofPCR. All reactions used BMB Expand HF polymerase mixture and conditionsspecified by the manufacturer. This PCR reaction resulted inamplification of a gene sequence of the correct size (1.1 kb). Theresulting fragment was digested with EcoR I and NheI and subcloned intothe corresponding sites of the plasmid pCI-neo. The nucleotide sequenceof the amplified fragment was determined by automated fluorescent DNAsequencing.

Cloning of construct IgE-2 (SEQ ID NO: 25).

The insert in construct IgE-2 consists of the signal sequence-human CH2domain followed by the human CH3/dog domain, followed by the human CH4domain. Assembly of the insert for IgE-2 consisted of using the signalsequence-human CH2 domain as a template for one PCR reaction, and thehuman CH4 domain as a template for a second PCR reaction. In these tworeactions, terminal primers were used to generate regions of homologywith the human CH3/dog CH3 domain, so that the two ends of the humanCH3/dog CH3 domain DNA fragment would hybridize to the two human domainDNA fragments and the three fragments would serve as “megaprimers” in aPCR reaction. The human CH3/dog CH3 domain was engineered to containoverlapping sequence on either end to the two human domain fragments(CH2 homology on the 5′ end and CH4 homology on the 3′ end). Then, thethree PCR fragments (human CH2, human CH3/dog CH3, human CH4) were mixedin a final PCR reaction utilizing two terminal primers to generate afull length product. The procedure is outlined as follows: Fragment 1:(signal sequence-human CH2 domain) 414 bp fragment resulting fromamplification of human CH2 domain with primers P171-S1b and P174-A404;fragment was amplified in 25 cycles of PCR, followed by getpurification. All reactions used BMB Expand HP polymerase mixture andconditions specified by the manufacturer. Fragment 2: (human CH3/dog CH3domain) 384 bp fragment described above; gel purified Fragment 3: (humanCH4 domain) 340 bp fragment resulting from amplification of human CH4domain with primers P174-S721 and P172-A1b; fragment was amplified in 25cycles of PCR, followed by gel purification. All reactions used BMBExpand HF polymerase mixture and conditions specified by themanufacturer. The three gel-purified fragments were added inapproximately equimolar amounts to a final PCR reaction using the twoterminal primers P171-S1b and P172-A1b, and carrying out 35 cycles ofPCR. All reactions used BMB Expand HF polymerase mixture and conditionsspecified by the manufacturer. This PCR reaction resulted inamplification of a gene sequence of the correct size (1.1 kb). Theresulting fragment was digested with EcoR I and Nhe I and subcloned intothe corresponding sites of the plasmid pCI-neo. The nucleotide sequenceof the amplified fragment was determined by automated fluorescent DNAsequencing.

Cloning of IgE-3 vaccine construct (SEQ ID NO: 26).

The insert in construct IgE-3 consists of the signal sequence-human CH2domain followed by the human CH3/conserved dog CH3 sequence, followed bythe human CH4domain. Assembly of the insert for IgE-3 consisted of usingthe signal sequence-human CH2domain as a template for one PCR reaction,and the human CH4 domain as a template for a second PCR reaction. Inthese two reactions, terminal primers were used to generate regions ofhomology with the human CH3/conserved dog CH3 sequence, so that the twoends of the middle (human CH3/conserved dog CH3 domain) DNA fragmentwould hybridize to the two human domain DNA fragments and the threefragments would serve as “megaprimers” in a PCR reaction. The humanCH3/conserved dog CH3 sequence was engineered to contain overlappingsequence on either end to the two human domain fragments (CH2 homologyon the 5′ end and CH4 homology on the 3′ end). Then, the three PCRfragments (human CH2, human CH3/conserved dog CH3, human CH4) were mixedin a final PCR reaction utilizing two terminal primers to generate afull length product The procedure is outlined as follows: Fragment 1:(signal sequence-human CH2 domain) 414 bp fragment resulting fromamplification of human CH2 domain with primers P171-S1b and P175-A404;fragment was amplified in 25 cycles of PCR, followed by gelpurification. All reactions used BMB Expand HF polymerase mixture andconditions specified by the manufacturer. Fragment 2: (humanCH3/conserved dog CH3 sequence) 384 bp fragment described above; gelpurified Fragment 3: (human CH4 domain) 340 bp fragment resulting fromamplification of human CH4 domain with primers P175-S715 and P172-A1b;fragment was amplified in 25 cycles of PCR, followed by gelpurification. All reactions used BMB Expand HF polymerase mixture andconditions specified by the manufacturer. The three gel-purifiedfragments were added in approximately equimolar amounts to a final PCRreaction using the two terminal primers P171-S1b and P172-A1b, andcarrying out 35 cycles of PCR. All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. ThisPCR reaction resulted in amplification of a gene sequence of the correctsize (1.1 kb). The resulting fragment was digested with EcoR 1 and Nhe Iand subcloned into the corresponding sites of the plasmid pCI-neo. Thenucleotide sequence of the amplified fragment was determined byautomated fluorescent DNA sequencing.

Cloning of IgE-4 vaccine construct (SEQ ID NO: 27).

The insert in construct IgE-4 consists of the signal sequence-human CH2domain followed by the human CH3 domain followed by the human CH4domain. Assembly of the insert for IgE-4 consisted of using the signalsequence-human CH2 domain as a template for one PCR reaction, and thehuman CH4 domain as a template for a second PCR reaction. In these tworeactions, terminal primers were used to generate regions of homologywith the human CH3 domain, so that the two ends of the middle (human CH3domain) DNA fragment would hybridize to the two terminal human domainDNA fragments and the three fragments would serve as “megaprimers” in aPCR reaction. The human CH3 domain sequence was engineered to containoverlapping sequence on either end to the two human domain fragments(CH2 homology on the 5′ end and CH4 homology on the 3″ end). Then, thethree PCR fragments (human CH2, human CH3, human CH4) were mixed in afinal PCR reaction utilizing two terminal primers to generate a fulllength product. The procedure is outlined as follows: Fragment 1:(signal sequence-human CH2 domain) 414 bp fragment resulting fromamplification of human CH2 domain with primers P171-S1b and P176-A404;fragment was amplified in 25 cycles of PCR, followed by gelpurification. All reactions used BMB Expand HF polymerase mixture andconditions specified by the manufacturer. Fragment 2: (human CH3domain)384 bp fragment described above; gel purified Fragment 3: (human CH4domain) 345 bp fragment resulting from amplification of human CH4 domainwith primers P176-S710and P172-A1b; fragment was amplified in 25 cyclesof PCR, followed by gel purification. All reactions used BMB Expand HFpolymerase mixture and conditions specified by the manufacturer. Thethree gel-purified fragments were added in approximately equimolaramounts to a final PCR reaction using the two terminal primers P171-S1band P172-A1b, and carrying out 35 cycles of PCR. All reactions used BMBExpand HF polymerase mixture and conditions specified by themanufacturer. This PCR reaction resulted in amplification of a genesequence of the correct size (1.1 kb). The resulting fragment wasdigested with EcoR I and Nhe I and subcloned into the correspondingsites of the plasmid pCI-neo. The nucleotide sequence of the amplifiedfragment was determined by automated fluorescent DNA sequencing.

Cloning of IgE-5 vaccine construct (SEQ ID NO: 28).

The insert in construct IgE-5 consists of the signal sequence-human CH2domain followed by the rat CH3 domain followed by the human CH4 domain.Assembly of the insert for IgE-5 consisted of using the signalsequence-human CH2 domain as a template for one PCR reaction, and thehuman CH4 domain as a template for a second PCR reaction. In these tworeactions, terminal primers were used to generate regions of homologywith the rat CH3 domain, so that the two ends of the middle (rat CH3domain) DNA fragment would hybridize to the two terminal human domainDNA fragments and the three fragments would serve as “megaprimers” in aPCR reaction. The rat CH3 domain sequence was engineered to containoverlapping sequence on either end to the two human domain fragments(CH2homology on the 5′ end and CH4 homology on the 3′ end). Then, thethree PCR fragments (human CH2, rat CH3, human CH4) were mixed in afinal PCR reaction utilizing two terminal primers to generate afull-length product. The procedure is outlined as follows: Fragment 1:(signal sequence-human CH2 domain) 411 bp fragment resulting fromamplification of human CH2 domain with primers P171-S1b and P177-A401;fragment was amplified in 25 cycles of PCR, followed by gelpurification. All reactions used BMB Expand HF polymerase mixture andconditions specified by the manufacturer. Fragment 2: (rat CH3 domain)384 bp fragment described above; gel purified Fragment 3: (human CH4domain) 341 bp fragment resulting from amplification of human CH4 domain(IgE-6(3′)) with primers P177-S711 and P172-A1b; fragment was amplifiedin 25 cycles of PCR, followed by gel purification. All reactions usedBMB Expand HF polymerase mixture and conditions specified by themanufacturer. The three gel-purified fragments were added inapproximately equimolar amounts to a final PCR reaction using the twoterminal primers P171-S1b and P172-A1b, and carrying out 35 cycles ofPCR. Alt reactions used BMB Expand HF polymerase mixture and conditionsspecified by the manufacturer. This PCR reaction resulted inamplification of a gene sequence of the correct size (1.1 kb). Theresulting fragment was digested with EcoR I and Nhe I and subcloned intothe corresponding sites of the plasmid pCI-neo. The nucleotide sequenceof the amplified fragment was determined by automated fluorescent DNAsequencing.

4. Transfection, expression and Reactivity of vaccines with anti-canineIgE antibodies.

Expression of IgE CH3 domain in insect cells:

Sf9 cells (Life Technologies) derived from Spodoptera frugiperda weretransfected with the Recombinant Bacmid DNA using Cell FECTIN reagent(Life Technologies) following the Bac-To-Bac Expression System protocol.At 72 hours supernates were passaged to fresh subconfluent Sf9 cells. At7 days post infection cytopathic effect (CPE) was evident. Supernateswere harvested and stored at 4 C protected from light. Samples wereanalyzed by electrophoresis (4-12% Bis-Tris Novex NuPage system reducingconditions). One of the duplicate gels was stained with coomassie blueand the other was transferred to PVDF membrane (Novex) using standardwestern blot transfer method. The membrane was probed with rabbit #145RBS-2 polyclonal antiserum followed by AP-rec Protein G (Zymed). Adistinct band of approximately 14.5 kDa was evident in both thecoomassie stained gel (FIG. 1) and western blot (FIG. 2) indicatingexpression of the CH3 protein.

Sequences of the present invention are described in Table 9.

TABLE 9 Sequence Listings Protein/ Composition Sequence ID# DNA Dog CH3domain SEQ ID NO: 1 Protein Human/dog CH3 domain fusion SEQ ID NO: 2Protein Human/dog CH3 domain chimera SEQ ID NO: 3 protein Human CH3domain SEQ ID NO: 4 Protein Rat CH3 domain SEQ ID NO: 5 Protein HumanCH3 domain (baculovirus expressed) SEQ ID NO: 6 protein Modified humanCH2 domain SEQ ID NO: 7 Protein Modified human CH4 domain SEQ ID NO: 8Protein human CH2—CH4 carrier protein SEQ ID NO: 9 protein IgE-1 fusionprotein SEQ ID NO: 10 Protein IgE-2 fusion protein SEQ ID NO: 11 ProteinIgE-3 fusion protein SEQ ID NO: 12 protein IgE-4 fusion protein SEQ IDNO: 13 Protein IgE-5 fusion protein SEQ ID NO: 14 protein Dog CH3 domainSEQ ID NO: 15 DNA Human/dog CH3 domain fusion SEQ ID NO: 16 DNAHuman/dog CH3 domain chimera SEQ ID NO: 17 DNA Human CH3 domain SEQ IDNO: 18 DNA Rat CH3 domain SEQ ID NO: 19 DNA Human CH3 domain(baculovirus SEQ ID NO: 20 DNA expressed) Modified human CH2 domain SEQID NO: 21 DNA Modified human CH4 domain SEQ ID NO: 22 DNA Modified humanCH2—CH4 carrier protein SEQ ID NO: 23 DNA IgE-1 construct SEQ ID NO: 24DNA IgE-2 construct SEQ ID NO: 25 DNA IgE-3 construct SEQ ID NO: 26 DNAIgE-4 construct SEQ ID NO: 27 DNA IgE-5 construct SEQ ID NO: 28 DNA

1. An isolated antigenic peptide comprising: (i) amino acid residues ofa CH3domain of an IgE molecule from a first species; (ii) amino acidresidues of a CH3 domain of an IgE molecule of a second unrelatedspecies, wherein the amino acid residues of the CH3 domain of the IgEmolecule from the first species are conserved in the CH3 domain of theIgE molecule of the second species, the amino acid residues of the CH3domain of the IgE molecule from the second species are not conserved inthe CH3 domain of the IgE molecule of the first species, and theantigenic peptide induces an anti-IgE immune response that does notcause anaphylaxis when administered to an animal.
 2. The antigenicpeptide of claim 1 comprising at least 10 amino acid residues of the CH3domain of the IgE molecule from either the first or second species. 3.The antigenic peptide of claim 1 comprising at least 10 amino acidresidues of the CH3 domain of the IgE molecule from the first speciesand at least 10 amino acid residues of the CH3 domain of the IgEmolecule from the second species.
 4. The antigenic peptide of claim 1comprising a total of between about 28 and about 31 amino acid residuesof the CH3 domain of the IgE molecule from the first species and the CH3domain of the IgE molecule from the second species. 5.-6. (canceled) 7.An isolated antigenic fusion protein comprising amino acid residues of aCH3 domain of an IgE molecule from a first species flanked by amino acidresidues of a CH3domain of an IgE molecule of a second unrelatedspecies, wherein the amino acid residues of the CH3 domain of the IgEmolecule from the first species are conserved in the CH3 domain of theIgE molecule of the second species, the amino acid residues of the CH3domain of the IgE molecule from the second species are not conserved inthe CH3 domain of the IgE molecule of the first species, and aheterologous protein carrier, wherein the antigenic fusion proteininduces an anti-IgE immune response that does not cause anaphylaxis whenadministered to an animal.
 8. (canceled)
 9. An isolated peptidecomprising an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQID NO:
 9. 10. The isolated antigenic fusion protein of claim 7 whereinthe heterologous protein carrier comprises the amino acid sequence ofSEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:
 9. 11-18. (canceled)
 19. Apharmaceutical composition for inducing an anti-IgE immune response thatdoes not cause anaphylaxis, comprising one or more antigenic peptideshaving an amino acid sequence comprising amino acid residues of a CH3domain of an IgE molecule or a fragment thereof species and apharmaceutically acceptable carrier.
 20. The pharmaceutical compositionof claim 19, wherein at least one antigenic peptide has the amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, or SEQ ID NO:
 14. 21. A pharmaceutical composition forinducing an anti-IgE immune response that does not cause anaphylaxiscomprising one or more antigenic fusion proteins having an amino acidsequence comprising amino acid residues of a CH3 domain of an IgEmolecule or a fragment thereof, a heterologous carrier protein and apharmaceutically acceptable carrier.
 22. The pharmaceutical compositionof claim 20, wherein at least one antigenic fusion protein has the aminoacid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO:
 14. 23.The pharmaceutical composition of claim 21, wherein the heterologouscarrier protein is selected from the group consisting of KLH, PhoE,rmLT, TraT, gD from BHV-1 virus, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ IDNO:
 9. 24. The pharmaceutical composition of claim 20, furthercomprising an adjuvant.
 25. The pharmaceutical composition of claim 20,wherein the anti-IgE immune response is the production of anti-IgEantibodies which bind to soluble IgE in serum and other bodily fluids,prevent IgE from binding to its high affinity receptors on mast cellsand basophils, and do not cross-link receptor-bound IgE. 26.-42.(canceled)
 43. The pharmaceutical composition of claim 21, furthercomprising an adjuvant.